Inhibition of mypb-c binding to myosin as a treatment for heart failure

ABSTRACT

The present invention provides for methods of treating and slowing the onset of heart failure. The inventors have determined that myosin binding to unphosphorylated Myosin Binding Protein C (MyBP-C) plays a key role in the diminution of cardiac contractile force and frequency in heart failure. The present invention provides peptide inhibitors of the MyBP-C/myosin interaction, thereby increasing both cardiac contractile force and frequency in the failing heart, as well as in patients not yet exhibiting frank heart failure.

This application claims benefit of priority to U.S. ProvisionalApplication 61/663,200, filed Jun. 22, 2012, the entire contents ofwhich are hereby incorporated by reference.

This invention was made with government support under HL082900 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of biology andmedicine. More particularly, it concerns molecular interactions betweenMyosin Binding Protein C (MyBP-C) and myosin in cardiac muscle.Specifically, the invention relates to the use of inhibitors of theinteraction between MyBP-C and myosin to increase the force of cardiacmuscle contraction and the rate of pressure development in the heart.

2. Description of Related Art

Heart failure is one of the leading causes of morbidity and mortality inthe world. In the U.S. alone, estimates indicate that 3 million peopleare currently living with cardiomyopathy and another 400,000 arediagnosed on a yearly basis. Dilated cardiomyopathy (DCM), also referredto as “congestive cardiomyopathy,” is the most common form of thecardiomyopathies and has an estimated prevalence of nearly 40 per100,000 individuals (Durand et al., 1995). Approximately half of DCMcases are idiopathic, and of these, familial dilated cardiomyopathy hasbeen indicated as representing approximately 20%. The remaining half ofDCM cases are associated with known disease processes, such as untreatedhypertension or valvular heart disease, as an end-stage condition.Furthermore, serious myocardial damage can result from certain drugsused in cancer chemotherapy (e.g., doxorubicin and daunoribucin). Inaddition, many DCM patients are chronic alcoholics. Fortunately, forthese patients, the progression of myocardial dysfunction may be stoppedor reversed if alcohol consumption is reduced or stopped early in thecourse of disease. Peripartum cardiomyopathy is another idiopathic formof DCM, as is disease associated with infectious sequelae. In sum,cardiomyopathies, including inherited or acquired DCM, are significantpublic health problems.

Heart disease and its manifestations, including coronary artery disease,myocardial infarction, congestive heart failure and cardiac hypertrophy,clearly present a major health risk in the United States today. The costto diagnose, treat and support patients suffering from these diseases iswell into the billions of dollars. Two particularly severemanifestations of heart disease are myocardial infarction and cardiachypertrophy. With respect to myocardial infarction, typically an acutethrombocytic coronary occlusion occurs in a coronary artery as a resultof atherosclerosis and causes myocardial cell death. Becausecardiomyocytes, the heart muscle cells, are terminally differentiatedand generally incapable of cell division, they are typically replaced byscar tissue when they die during the course of an acute myocardialinfarction. Scar tissue is not contractile, fails to contribute tocardiac function, and often plays a detrimental role in heart functionby expanding during cardiac contraction, or by increasing the size andeffective radius of the ventricle, for example, becoming hypertrophic.With respect to non-physiological (i.e., not due to exercise training)cardiac hypertrophy, one theory regards this as a disease that resemblesaberrant development and, as such, raises the question of whetherdevelopmental signals in the heart can contribute to hypertrophicdisease. Cardiac hypertrophy is an adaptive response of the heart tovirtually all forms of cardiac disease, including those arising fromhypertension, mechanical load, myocardial infarction, cardiacarrhythmias, endocrine disorders, and genetic mutations in cardiaccontractile protein genes. While the hypertrophic response is initiallya compensatory mechanism that augments cardiac output, sustainedhypertrophy can lead to DCM, heart failure, and sudden death. In theUnited States, approximately half a million individuals are diagnosedwith heart failure each year, with a five-year mortality rateapproaching 50%.

The causes and effects of cardiac hypertrophy have been extensivelydocumented, but the underlying molecular mechanisms have not beenelucidated. Understanding these mechanisms is a major concern in theprevention and treatment of cardiac disease and will be crucial as atherapeutic modality in designing new drugs that specifically targetcardiac hypertrophy and subsequent heart failure. As pathologic cardiachypertrophy most often does not produce any symptoms until the cardiacdamage is severe enough to produce heart failure, the symptoms ofcardiomyopathy are those associated with heart failure. These symptomsinclude shortness of breath, fatigue with exertion, the inability to lieflat without becoming short of breath (orthopnea), paroxysmal nocturnaldyspnea, enlarged cardiac dimensions, and/or swelling in the lower legs.Patients also often present with increased blood pressure, extra heartsounds, cardiac murmurs, pulmonary and systemic emboli, chest pain,pulmonary congestion, and palpitations. In addition, DCM causesdecreased ejection fractions (i.e., a measure of both intrinsic systolicfunction and remodeling). The disease is further characterized byventricular dilation and grossly impaired systolic function due todiminished myocardial contractility, which results in dilated heartfailure in many patients. Affected hearts also undergo cell/chamberremodeling as a result of the myocyte/myocardial dysfunction, whichcontributes to the “DCM phenotype.” As the disease progresses so do thesymptoms. Patients with DCM also have a greatly increased incidence oflife-threatening arrhythmias, including ventricular tachycardia andventricular fibrillation. In these patients, an episode of syncope(dizziness) is regarded as a harbinger of sudden death.

Diagnosis of dilated cardiomyopathy typically depends upon thedemonstration of enlarged heart chambers, particularly enlargedventricles. Enlargement is commonly observable on chest X-rays, but ismore accurately assessed using echocardiograms. DCM is often difficultto distinguish from acute myocarditis, valvular heart disease, coronaryartery disease, and hypertensive heart disease. Once the diagnosis ofdilated cardiomyopathy is made, every effort is made to identify andtreat potentially reversible causes and prevent further heart damage.For example, coronary artery disease and valvular heart disease must beruled out. Anemia, abnormal tachycardias, nutritional deficiencies,alcoholism, thyroid disease and/or other problems need to be addressedand controlled.

As mentioned above, treatment with pharmacological agents stillrepresents the primary mechanism for reducing or eliminating themanifestations of heart failure. Diuretics constitute the first line oftreatment for mild-to-moderate heart failure by reducing the volume loadon the heart. Unfortunately, many of the commonly used diuretics (e.g.,the thiazides) have numerous adverse effects. For example, certaindiuretics may increase serum cholesterol and triglycerides. Moreover,diuretics are generally ineffective for patients suffering from severeheart failure. If diuretics are ineffective, vasodilatory agents may beused. The angiotensin converting (ACE) inhibitors (e.g., enalopril andlisinopril) not only provide symptomatic relief, they also have beenreported to decrease mortality (Young et al., 1989). Again, however, theACE inhibitors are associated with adverse effects that result in theirbeing contraindicated in patients with certain disease states (e.g.,renal artery stenosis). Similarly, inotropic agent therapy (i.e., a drugthat improves cardiac output by increasing the force of myocardialmuscle contraction) is associated with a panoply of adverse reactions,including gastrointestinal problems and central nervous systemdysfunction. Thus, the currently used pharmacological agents have severeshortcomings in particular patient populations. The availability of new,safe and effective agents would undoubtedly benefit patients who eithercannot use the pharmacological modalities presently available, or who donot receive adequate relief from those modalities.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided amethod of treating heart failure comprising (a) identifying a patientexhibiting one or more symptoms of heart failure; and (b) administeringto said patient an inhibitor of the interaction of Myosin BindingProtein C (MyBP-C) with myosin. The inhibitor may be a peptide derivedfrom the MyBP-C binding site for myosin. Such a peptide may be of nomore than 50 residues and comprise the sequence LKKRDXFRRD (SEQ ID NO:1), where X is A, V or D. The peptide may also comprise, consistessentially of or consist of the sequence FSSLLKKRDXFRRD (SEQ ID NO:48), FSSLLKKRDXFRRDXK (SEQ ID NO: 49), LKKRDXFRRDXKLE (SEQ ID NO: 50),SLLKKRDXFRRDXKLE (SEQ ID NO: 51), or FSSLLKKRDXFRRDXKLE (SEQ ID NO: 52),and optionally may be no more than 25, 30, 35, 40 or 45 residues. Thepeptide may comprise a cell penetrating domain. The peptide may comprisesome or all D-amino acids, such as an all D-amino acid peptide in aretro-inverso configuration.

Administering the inhibitor may be performed intramuscularly,intravenously or by direct injection into cardiac tissue, or maycomprise oral, transdermal, sustained release, controlled release,delayed release, suppository, or sublingual administration. The methodmay further comprise administering to said patient a second heartfailure therapy. The second therapy may be selected from the groupconsisting of a beta blocker, an ionotrope, a diuretic, ACE-I, AIIantagonist, BNP, or a Ca⁺⁺ channel blocker. The second therapy may beadministered at the same time as said inhibitor of the interaction ofMyBP-C and myosin or either before or after said inhibitor of theinteraction of MyBP-C and myosin.

Treating may comprise improving one or more symptoms of heart failure,such as increased exercise capacity, increased cardiac ejection volume,increased cardiac ejection fraction, decreased left ventricular enddiastolic pressure, decreased pulmonary capillary wedge pressure,increased cardiac output, or cardiac index, lowered pulmonary arterypressures, decreased left ventricular end systolic and diastolicdimensions, decreased left and right ventricular wall stress, decreasedwall tension, increased quality of life, and decreased disease-relatedmorbidity or mortality.

In another embodiment, there is provided a method of slowing theprogression of heart failure comprising (a) identifying a patient atrisk of developing severe heart failure; and (b) administering to saidpatient an inhibitor of the interaction of Myosin Binding Protein C(MyBP-C) and myosin. The inhibitor may be a peptide derived from theMyBP-C binding site for myosin. Such a peptide may be of no more than 50residues and comprise the sequence LKKRDXFRRD (SEQ ID NO: 1), where X isA, V or D. The peptide may also comprise, consist essentially of orconsist of the sequence FSSLLKKRDXFRRD (SEQ ID NO: 48), FSSLLKKRDXFRRDXK(SEQ ID NO: 49), LKKRDXFRRDXKLE (SEQ ID NO: 50), SLLKKRDXFRRDXKLE (SEQID NO: 51), or FSSLLKKRDXFRRDXKLE (SEQ ID NO: 52), and optionally may beno more than 25, 30, 35, 40 or 45 residues. The peptide may comprise acell penetrating domain. The peptide may comprise some or all D-aminoacids, such as an all D-amino acid peptide in a retro-inversoconfiguration.

Administering may be performed intramuscularly, intravenously or bydirect injection into cardiac tissue, or may comprise oral, transdermal,sustained release, controlled release, delayed release, suppository, orsublingual administration. The patient at risk may exhibit one or moreof a list of risk factors comprising long-standing uncontrolledhypertension, uncorrected valvular disease, chronic angina, recentmyocardial infarction, congenital predisposition to heart disease orpathological hypertrophy. The patient at risk may be diagnosed as havinga genetic predisposition to heart failure, and/or have a familialhistory of heart failure. The method may further comprise administeringto said patient a second heart failure therapy, such as a beta blocker,an ionotrope, a diuretic, ACE-I, AII antagonist, BNP, or a Ca⁺⁺ channelblocker. The subject may be a heart transplant recipient.

In other embodiments, there are provided:

a method of increasing exercise tolerance in a subject with heartfailure comprising administering to said subject an inhibitor of theinteraction of Myosin Binding Protein C (MyBP-C) and myosin;

a method of reducing hospitalization in a subject with heart failurecomprising administering to said subject an inhibitor of the interactionof Myosin Binding Protein C (MyBP-C) and myosin;

a method of improving quality of life in a subject with heart failurecomprising administering to said subject an inhibitor of the interactionof Myosin Binding Protein C (MyBP-C) and myosin;

a method of decreasing morbidity in a subject with heart failurecomprising administering to said subject an inhibitor of the interactionof Myosin Binding Protein C (MyBP-C) and myosin; or

a method of decreasing mortality in a subject with heart failurecomprising administering to said patient an inhibitor of the interactionof Myosin Binding Protein C (MyBP-C) and myosin.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The word “about” means plus or minus 5% ofthe stated number.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Force pCa relationships obtained in absence or presence ofpeptide. Left panel: The force-pCa relationship was shifted to lowerCa²⁺ concentrations by infusion of the 302a peptide. Circles are controldata before peptide; triangles are experimental data following peptidetreatment. Right panel: The force-pCa relationship was unaffected bytreatment with a scrambled peptide. Circles are control data beforepeptide; diamonds are experimental data following peptide treatment.

FIG. 2. Effects of peptides on the rate of force development in murinemyocardium. Left panel: The rate constant of force development (ktr) wasincreased at each pCa studied by treatment of skinned myocardium with 50μM 302a. Circles are control data before peptide; triangles are datafollowing treatment with peptide 302a. Right panel: The rate of forcedevelopment was unaffected by treatment with a scrambled peptide.Circles are control data before peptide; diamonds are control datafollowing peptide treatment.

FIG. 3. Comparison of effects of peptide 302a on the Ca²⁺ sensitivity offorce in murine and porcine myocardium. Left panel: Treatment of mousemyocardium with 50 μM 302a increased the Ca²⁺ sensitivity of force by0.15 pCa units, i.e., pCa₅₀ was 5.70±0.02 in control myocardium and5.85±0.02 following treatment with peptide. Right panel: Treatment ofporcine myocardium with 50 μM 302a increased the Ca²⁺ sensitivity offorce by 0.32 pCa units, i.e., pCa₅₀ was 5.60 in control myocardium and5.92 following treatment with peptide. Washout of the peptide reversedthe effect.

FIG. 4. Peptide variants.

FIG. 5. Non-effect of scrambled or phosphomimetic (A→D) 18-mer peptides.

FIG. 6. Relative effects of various peptides on force at pCa 5.9 (scaledto force at pCa 4.5), mouse.

FIG. 7. Relative effects of various peptides on force at pCa 5.9 (scaledto force at pCa 4.5), mouse.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Heart failure is generally characterized as an inability of the heart topump adequate amounts of blood throughout the body. Heart attacks, heartdisease, and hypertension can all lead to heart failure. It is estimatedthat 2% of adults in developed countries suffer from heart failure andthat number increases to 6-10% of people over the age of 65. To overcomethe symptoms of heart failure, pharmaceuticals that improve the forceand number of heart contractions are needed. The present inventionprovides a novel approach to achieving increased force and power ofcardiac muscle contractions using new therapeutic compositions. Theseare discussed in detail below.

I. MYOSIN

Myosins comprise a family of ATP-dependent motor proteins and are bestknown for their role in muscle contraction and their involvement in awide range of other eukaryotic motility processes. They are responsiblefor actin-based motility. The term was originally used to describe agroup of similar ATPases found in striated and smooth muscle cells.Thus, although myosin was originally thought to be restricted to musclecells, there is no single “myosin” but rather a huge superfamily ofgenes whose protein products share the basic properties of actinbinding, ATP hydrolysis (ATPase enzyme activity), and forcetransduction. Virtually all eukaryotic cells contain myosin isoforms.Some isoforms have specialized functions in certain cell types (such asmuscle), while other isoforms are ubiquitous. The structure and functionof myosin is strongly conserved across species, to the extent thatrabbit muscle myosin II will bind to actin from an amoeba.

Most myosin molecules are composed of a head, neck, and tail domain. Thehead domain binds the filamentous actin, and uses ATP hydrolysis togenerate force and to “walk” along the filament towards the barbed (+)end (with the exception of myosin VI, which moves towards the pointed(−) end). The neck domain acts as a linker and as a lever arm fortransducing force generated by the catalytic motor domain. The neckdomain can also serve as a binding site for myosin light chains whichare distinct proteins that form part of a macromolecular complex andgenerally have regulatory functions. The tail domain generally mediatesinteraction with cargo molecules and/or other myosin subunits. In somecases, the tail domain may play a role in regulating motor activity.

Multiple myosin II molecules generate force in skeletal muscle through apower stroke mechanism fuelled by the energy released from ATPhydrolysis. The power stroke occurs at the release of phosphate from themyosin molecule after the ATP hydrolysis while myosin is tightly boundto actin. The effect of this release is a conformational change in themolecule that pulls against the actin. The release of the ADP moleculeand binding of a new ATP molecule will release myosin from actin. ATPhydrolysis within the myosin will cause it to bind to actin again torepeat the cycle. The combined effect of the myriad power strokes causesthe muscle to contract.

Myosin II (also known as conventional myosin) is the myosin typeresponsible for producing muscle contraction in muscle cells. Myosin IIcontains two heavy chains, each about 2000 amino acids in length, whichconstitute the head and tail domains. Each of these heavy chainscontains the N-terminal head domain, while the C-terminal tails take ona coiled-coil morphology, holding the two heavy chains together (imaginetwo snakes wrapped around each other, such as in a caduceus). Thus,myosin II has two heads. The intermediate neck domain is the regioncreating the angle between the head and tail. In smooth muscle, a singlegene (MYH11) codes for the heavy chains myosin II, but splice variantsof this gene result in four distinct isoforms. It also contains 4 lightchains, resulting in 2 per head, weighing 20 (MLC20) and 17 (MLC17) kDa.These bind the heavy chains in the “neck” region between the head andtail. The MLC20 is also known as the regulatory light chain and activelyparticipates in muscle contraction. The MLC17 is also known as theessential light chain. Its exact function is unclear, but is believed tocontribute to the structural stability of the myosin, head along withMLC20. Two variants of MLC17 (MLC17a/b) exist as a result of alternatesplicing at the MLC17 gene.

In muscle cells, the long coiled-coil tails of the individual myosinmolecules join together, forming the thick filaments of the sarcomere.The force-producing head domains stick out from the side of the thickfilament, ready to walk along the adjacent actin-based thin filaments inresponse to the proper chemical signals.

II. MYOSIN BINDING PROTEIN C

Myosin binding protein C (MyBP-C) has been shown by this laboratory tobe a central regulator of the kinetics of cardiac contraction. In murinemodels, the inventors have observed in published work that geneticablation or phosphorylation of MyBP-C by PKA or CAMKII accelerates thekinetics of contraction and increases the force of contraction incardiac muscle. Most recently, the inventors have shown in unpublishedwork that CAMKII phosphorylation of MyBP-C at residues S282 and S302 inthe mouse and S284 and S304 in the human underlies the increase inmyocardial force of contraction as heart rate is increased, theso-called staircase phenomenon. Proof for this conclusion was obtainedby observing that (1) phosphorylation at these two residues is increasedwhen stimulus frequency is increased, but other potentialphosphorylation sites are not, and (2) the staircase phenomenon isabsent in hearts in which these residues are replaced withnon-phosphorylatable residues. In terms of molecular mechanism, theinventors believe that MyBP-C normally depresses the speed and strengthof contraction by means of its interaction with the contractile proteinmyosin and thereby reduces the probability of myosin binding to actin.Ablation of MyBP-C or phosphorylation of MyBP-C by PKA or CAMKIIdisrupts this interaction (we have evidence for this for PKA from theinventors published in vitro studies) and relieves the repression ofmyosin by MyBP-C (the inventors have evidence for this from theirpublished x-ray diffraction studies). Once phosphorylated, MyBP-C nolonger binds to myosin, myosin moves closer to actin, the probability ofmyosin binding to actin increases, and the speed and strength ofcontraction both increase.

In heart failure, MyBP-C is phosphorylated minimally or not at all byeither PKA or CAMKII due to down-regulation of β-adrenergic receptors.Based on the inventors' findings that phosphorylation of MyBP-C improvescontraction, the inventors propose to target the CAMKII site(s) onMyBP-C with a small-molecule pharmaceutical to disrupt its interactionwith myosin and thereby improve cardiac contraction and pump function.This approach is far superior to targeting PKA sites since there aremany fewer CAMKII phosphorylation sites in the heart, which improvesspecificity of targeting. By specifically targeting residues S282 and/orS302 in the mouse and S284 and/or S304 in the human, it should bepossible to minimize any increase in metabolic load on the heart thatmight be introduced by increased strength of beating, an increase thatwould not be tolerated well by a failing heart.

III. PEPTIDE INHIBITORS

A. Structure

The present invention contemplates the design, production and use ofvarious MyBP-C peptides. The structural features of these peptides areas follows. First, the peptides will generally have no more than 50consecutive residues of a MyBP-C. Thus, the term “a peptide having nomore than 50 consecutive residues,” even when including the term“comprising,” cannot be understood to comprise a greater number ofconsecutive MyBP-C residues. Second, the peptides will contain themotifs responsible for interaction with myosin. In general, the peptideswill have, at a minimum, 8 consecutive residues of the MyBP-C.

In general, the peptides will be 15-50 residues or less, again,comprising no more than 50 consecutive residues of a MyBP-C. The overalllength may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 residues. Ranges of peptidelength of 10-50 residues, 15-50 residues, 20-25 residues, 25-50residues, 30-50 residues, 35-50, residues, 10-20 residues, 15-20residues, 15-25 residues, 15-30 residues, 20-25 residues, and 20-25residues are contemplated. The number of consecutive MyBP-C residues maybe 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater. Rangesof consecutive residues of 15-20 residues, 20-25 residues, 15-30,residues, 20-30 residues or 15-25 residues are contemplated.

The present invention may utilize L-configuration amino acids,D-configuration amino acids, or a mixture thereof. While L-amino acidsrepresent the vast majority of amino acids found in proteins, D-aminoacids are found in some proteins produced by exotic sea-dwellingorganisms, such as cone snails. They are also abundant components of thepeptidoglycan cell walls of bacteria. D-serine may act as aneurotransmitter in the brain. The L and D convention for amino acidconfiguration refers not to the optical activity of the amino aciditself, but rather to the optical activity of the isomer ofglyceraldehyde from which that amino acid can theoretically besynthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde islevorotary).

One form of an “all-D” peptide is a retro-inverso peptide. Retro-inversomodification of naturally-occurring polypeptides involves the syntheticassemblage of amino acids with α-carbon stereochemistry opposite to thatof the corresponding L-amino acids, i.e., D-amino acids in reverse orderwith respect to the native peptide sequence. A retro-inverso analoguethus has reversed termini and reversed direction of peptide bonds (NH—COrather than CO—NH) while approximately maintaining the topology of theside chains as in the native peptide sequence. See U.S. Pat. No.6,261,569, incorporated herein by reference.

As mentioned above, the present invention contemplates fusing orconjugating a cell delivery domain (also called a cell delivery vector,or cell transduction domain). Such domains are well known in the art andare generally characterized as short amphipathic or cationic peptidesand peptide derivatives, often containing multiple lysine and arginineresides (Fischer, 2007). Of particular interest are poly-D-Arg andpoly-D-Lys sequences (e.g., dextrorotary residues, eight residues inlength), while others are shown in Table 1, below.

TABLE 1 SEQ ID CDD/CTD PEPTIDES NO QAATATRGRSAASRPTERPRAPARSASRPRRPVE  2RQIKIWFQNRRMKWKK  3 RRMKWKK  4 RRWRRWWRRWWRRWRR  5 RGGRLSYSRRRFSTSTGR  6YGRKKRRQRRR  7 RKKRRQRRR  8 YARAAARQARA  9 RRRRRRRR 10 KKKKKKKK 11GWTLNSAGYLLGKINLKALAALAKXIL 12 LLILLRRRIRKQANAHSK 13 SRRHHCRSKAKRSRHH 14NRARRNRRRVR 15 RQLRIAGRRLRGRSR 16 KLIKGRTPIKFGK 17 RRIPNRRPRR 18KLALKLALKALKAALKLA 19 KLAKLAKKLAKLAK 20 GALFLGFLGAAGSTNGAWSQPKKKRKV 21KETWWETWWTEWSQPKKKRKV 22 GALFLGWLGAAGSTMGAKKKRKV 23MGLGLHLLVLAAALQGAKSKRKV 24 AAVALLPAVLLALLAPAAANYKKPKL 25MANLGYWLLALFVTMWTDVGLCKKRPKP 26 LGTYTQDFNKFHTFPQTAIGVGAP 27DPKGDPKGVTVTVTVTVTGKGDPXPD 28 PPPPPPPPPPPPPP 29 VRLPPPVRLPPPVRLPPP 30PRPLPPPRPG 31 SVRRRPRPPYLPRPRPPPFFPPRLPPRIPP 32 TRSSRAGLQFPVGRVHRLLRK 33GIGKFLHSAKKFGKAFVGEIMNS 34 KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK 35ALWMTLLKKVLKAAAKAALNAVLVGANA 36 GIGAVLKVLTTGLPALISWIKRKRQQ 37INLKALAALAKKIL 38 GFFALIPKIISSPLPKTLLSAVGSALGGSGGQE 39 LAKWALKQGFAKLKS40 SMAQDIISTIGDLVKWIIQTVNXFTKK 41LLGDFFRKSKEKIGKEFKRIVQRIKQRIKDFLANLVPRTES 42 LKKLLKKLLKKLLKKLLKKL 43KLKLKLKLKLKLKLKLKL 44 PAWRKAFRWAWRMLKKAA 45Also as mentioned above, peptides modified for in vivo use by theaddition, at the amino- and/or carboxyl-terminal ends, of a blockingagent to facilitate survival of the peptide in vivo are contemplated.This can be useful in those situations in which the peptide termini tendto be degraded by proteases prior to cellular uptake. Such blockingagents can include, without limitation, additional related or unrelatedpeptide sequences that can be attached to the amino and/or carboxylterminal residues of the peptide to be administered. These agents can beadded either chemically during the synthesis of the peptide, or byrecombinant DNA technology by methods familiar in the art.Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino- and/orcarboxyl-terminal residues.

B. Synthesis

It will be advantageous to produce peptides using the solid-phasesynthetic techniques (Merrifield, 1963). Other peptide synthesistechniques are well known to those of skill in the art (Bodanszky etal., 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia,1984). Appropriate protective groups for use in such syntheses will befound in the above texts, as well as in Protective Groups in OrganicChemistry, 1973. These synthetic methods involve the sequential additionof one or more amino acid residues or suitable protected amino acidresidues to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group, such as lysine.

Using solid phase synthesis as an example, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptides of the invention are preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

Aside from the 20 standard amino acids can be used, there are a vastnumber of “non-standard” amino acids. Two of these can be specified bythe genetic code, but are rather rare in proteins. Selenocysteine isincorporated into some proteins at a UGA codon, which is normally a stopcodon. Pyrrolysine is used by some methanogenic archaea in enzymes thatthey use to produce methane. It is coded for with the codon UAG.Examples of non-standard amino acids that are not found in proteinsinclude lanthionine, 2-aminoisobutyric acid, dehydroalanine and theneurotransmitter gamma-aminobutyric acid. Non-standard amino acids oftenoccur as intermediates in the metabolic pathways for standard aminoacids—for example ornithine and citrulline occur in the urea cycle, partof amino acid catabolism. Non-standard amino acids are usually formedthrough modifications to standard amino acids. For example, homocysteineis formed through the transsulfuration pathway or by the demethylationof methionine via the intermediate metabolite S-adenosyl methionine,while hydroxyproline is made by a posttranslational modification ofproline.

C. Linkers

Linkers or cross-linking agents may be used to fuse MyBP-C peptides toother proteinaceous sequences. Bifunctional cross-linking reagents havebeen extensively used for a variety of purposes including preparation ofaffinity matrices, modification and stabilization of diverse structures,identification of ligand and receptor binding sites, and structuralstudies. Homobifunctional reagents that carry two identical functionalgroups proved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino-,sulfhydryl-, guanidino-, indole-, or carboxyl-specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis and the mildreaction conditions under which they can be applied. A majority ofheterobifunctional cross-linking reagents contains a primaryamine-reactive group and a thiol-reactive group.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described in U.S. Pat.No. 5,889,155, specifically incorporated herein by reference in itsentirety. The cross-linking reagents combine a nucleophilic hydrazideresidue with an electrophilic maleimide residue, allowing coupling inone example, of aldehydes to free thiols. The cross-linking reagent canbe modified to cross-link various functional groups and is thus usefulfor cross-linking polypeptides. In instances where a particular peptidedoes not contain a residue amenable for a given cross-linking reagent inits native sequence, conservative genetic or synthetic amino acidchanges in the primary sequence can be utilized.

Another use of linkers in the context of peptides as therapeutics is theso-called “Stapled Peptide” technology of Aileron Therapeutics. Thegeneral approach for “stapling” a peptide is that two key residueswithin the peptide are modified by attachment of linkers through theamino acid side chains. Once synthesized, the linkers are connectedthrough a catalyst, thereby creating a bridge the physically constrainsthe peptide into its native α-helical shape. In addition to helpingretain the native structure needed to interact with a target molecule,this conformation also provides stability against peptidases as well ascell-permeating properties. U.S. Pat. Nos. 7,192,713 and 7,183,059,describing this technology, are hereby incorporated by reference. Seealso Schafineister et al., 2000.

D. Design, Variants and Analogs

Having identified structures in MyBP-C interaction with myosin, theinventors also contemplate that variants of the sequences may beemployed. For example, certain non-natural amino acids that satisfy thestructural constraints of the sequences may be substituted without aloss, and perhaps with an improvement in, biological function. Inaddition, the present inventors also contemplate that structurallysimilar compounds may be formulated to mimic the key portions of peptideor polypeptides of the present invention. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and, hence, also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Methods for generating specific structures have been disclosed in theart. For example, α-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. Methods for generatingconformationally restricted β-turns and β-bulges are described, forexample, in U.S. Pat. Nos. 5,440,013; 5,618,914; and 5,670,155. Othertypes of mimetic turns include reverse and γ-turns. Reverse turnmimetics are disclosed in U.S. Pat. Nos. 5,475,085 and 5,929,237, andγ-turn mimetics are described in U.S. Pat. Nos. 5,672,681 and 5,674,976.

As used herein, “molecular modeling” means quantitative and/orqualitative analysis of the structure and function of protein-proteinphysical interaction based on three-dimensional structural informationand protein-protein interaction models. This includes conventionalnumeric-based molecular dynamic and energy minimization models,interactive computer graphic models, modified molecular mechanicsmodels, distance geometry and other structure-based constraint models.Molecular modeling typically is performed using a computer and may befurther optimized using known methods. Computer programs that use X-raycrystallography data are particularly useful for designing suchcompounds. Programs such as RasMol, for example, can be used to generatethree dimensional models. Computer programs such as INSIGHT (Accelrys,Burlington, Mass.), GRASP (Anthony Nicholls, Columbia University), Dock(Molecular Design Institute, University of California at San Francisco),and Auto-Dock (Accelrys) allow for further manipulation and the abilityto introduce new structures. The methods can involve the additional stepof outputting to an output device a model of the 3-D structure of thecompound. In addition, the 3-D data of candidate compounds can becompared to a computer database of, for example, 3-D structures.

Compounds of the invention also may be interactively designed fromstructural information of the compounds described herein using otherstructure-based design/modeling techniques (see, e.g., Jackson, 1997;Jones et al., 1996). Candidate compounds can then be tested in standardassays familiar to those skilled in the art. Exemplary assays aredescribed herein.

The 3-D structure of biological macromolecules (e.g., proteins, nucleicacids, carbohydrates, and lipids) can be determined from data obtainedby a variety of methodologies. These methodologies, which have beenapplied most effectively to the assessment of the 3-D structure ofproteins, include: (a) x-ray crystallography; (b) nuclear magneticresonance (NMR) spectroscopy; (c) analysis of physical distanceconstraints formed between defined sites on a macromolecule, e.g.,intramolecular chemical crosslinks between residues on a protein (e.g.,PCT/US00/14667, the disclosure of which is incorporated herein byreference in its entirety), and (d) molecular modeling methods based ona knowledge of the primary structure of a protein of interest, e.g.,homology modeling techniques, threading algorithms, or ab initiostructure modeling using computer programs such as MONSSTER (Modeling OfNew Structures from Secondary and Tertiary Restraints) (see, e.g.,International Application No. PCT/US99/11913, the disclosure of which isincorporated herein by reference in its entirety). Other molecularmodeling techniques may also be employed in accordance with thisinvention (e.g., Cohen et al., 1990; Navia et al., 1992), thedisclosures of which are incorporated herein by reference in theirentirety). All these methods produce data that are amenable to computeranalysis. Other spectroscopic methods that can also be useful in themethod of the invention, but that do not currently provide atomic levelstructural detail about biomolecules, include circular dichroism andfluorescence and ultraviolet/visible light absorbance spectroscopy. Apreferred method of analysis is x-ray crystallography. Descriptions ofthis procedure and of NMR spectroscopy are provided below.

X-ray Crystallography.

X-ray crystallography is based on the diffraction of x-radiation of acharacteristic wavelength by electron clouds surrounding the atomicnuclei in a crystal of a molecule or molecular complex of interest. Thetechnique uses crystals of purified biological macromolecules ormolecular complexes (but these frequently include solvent components,co-factors, substrates, or other ligands) to determine near atomicresolution of the atoms making up the particular biologicalmacromolecule. A prerequisite for solving 3-D structure by x-raycrystallography is a well-ordered crystal that will diffract x-raysstrongly. The method directs a beam of x-rays onto a regular, repeatingarray of many identical molecules so that the x-rays are diffracted fromthe array in a pattern from which the structure of an individualmolecule can be retrieved. Well-ordered crystals of, for example,globular protein molecules are large, spherical or ellipsoidal objectswith irregular surfaces. The crystals contain large channels between theindividual molecules. These channels, which normally occupy more thanone half the volume of the crystal, are filled with disordered solventmolecules, and the protein molecules are in contact with each other atonly a few small regions. This is one reason why structures of proteinsin crystals are generally the same as those of proteins in solution.

Methods of obtaining the proteins of interest are described below. Theformation of crystals is dependent on a number of different parameters,including pH, temperature, the concentration of the biologicalmacromolecule, the nature of the solvent and precipitant, as well as thepresence of added ions or ligands of the protein. Many routinecrystallization experiments may be needed to screen all these parametersfor the combinations that give a crystal suitable for x-ray diffractionanalysis. Crystallization robots can automate and speed up work ofreproducibly setting up a large number of crystallization experiments(see, e.g., U.S. Pat. No. 5,790,421, the disclosure of which isincorporated herein by reference in its entirety).

Polypeptide crystallization occurs in solutions in which the polypeptideconcentration exceeds its solubility maximum (i.e., the polypeptidesolution is supersaturated). Such solutions may be restored toequilibrium by reducing the polypeptide concentration, preferablythrough precipitation of the polypeptide crystals. Often polypeptidesmay be induced to crystallize from supersaturated solutions by addingagents that alter the polypeptide surface charges or perturb theinteraction between the polypeptide and bulk water to promoteassociations that lead to crystallization.

Crystallizations are generally carried out between 4° C. and 20° C.Substances known as “precipitants” are often used to decrease thesolubility of the polypeptide in a concentrated solution by forming anenergetically unfavorable precipitating depleted layer around thepolypeptide molecules (Weber, 1991). In addition to precipitants, othermaterials are sometimes added to the polypeptide crystallizationsolution. These include buffers to adjust the pH of the solution andsalts to reduce the solubility of the polypeptide. Various precipitantsare known in the art and include the following: ethanol, 3-ethyl-2-4pentanediol, and many of the polyglycols, such as polyethylene glycol(PEG). The precipitating solutions can include, for example, 13-24% PEG4000, 5-41% ammonium sulfate, and 1.0-1.5 M sodium chloride, and a pHranging from 5.0-7.5. Other additives can include 0.1M Hepes, 2-4%butanol, 20-100 mM sodium acetate, 50-70 mM citric acid, 120-130 mMsodium phosphate, 1 mM ethylene diamine tetraacetic acid (EDTA), and 1mM dithiothreitol (DTT). These agents are prepared in buffers and areadded dropwise in various combinations to the crystallization buffer.Proteins to be crystallized can be modified, e.g., by phosphorylation orby using a phosphate mimic (e.g., tungstate, cacodylate, or sulfate).

Commonly used polypeptide crystallization methods include the followingtechniques: batch, hanging drop, seed initiation, and dialysis. In eachof these methods, it is important to promote continued crystallizationafter nucleation by maintaining a supersaturated solution. In the batchmethod, polypeptide is mixed with precipitants to achievesupersaturation, and the vessel is sealed and set aside until crystalsappear. In the dialysis method, polypeptide is retained in a sealeddialysis membrane that is placed into a solution containing precipitant.Equilibration across the membrane increases the polypeptide andprecipitant concentrations, thereby causing the polypeptide to reachsupersaturation levels.

In the hanging drop technique (McPherson, 1976), an initial polypeptidemixture is created by adding a precipitant to a concentrated polypeptidesolution. The concentrations of the polypeptide and precipitants aresuch that in this initial form, the polypeptide does not crystallize. Asmall drop of this mixture is placed on a glass slide that is invertedand suspended over a reservoir of a second solution. The system is thensealed. Typically, the second solution contains a higher concentrationof precipitant or other dehydrating agent. The difference in theprecipitant concentrations causes the protein solution to have a highervapor pressure than the second solution. Since the system containing thetwo solutions is sealed, an equilibrium is established, and water fromthe polypeptide mixture transfers to the second solution. Thisequilibrium increases the polypeptide and precipitant concentration inthe polypeptide solution. At the critical concentration of polypeptideand precipitant, a crystal of the polypeptide may form.

Another method of crystallization introduces a nucleation site into aconcentrated polypeptide solution. Generally, a concentrated polypeptidesolution is prepared and a seed crystal of the polypeptide is introducedinto this solution. If the concentrations of the polypeptide and anyprecipitants are correct, the seed crystal will provide a nucleationsite around which a larger crystal forms.

Yet another method of crystallization is an electrocrystallizationmethod in which use is made of the dipole moments of proteinmacromolecules that self-align in the Helmholtz layer adjacent to anelectrode (see, e.g., U.S. Pat. No. 5,597,457, the disclosure of whichis incorporated herein by reference in its entirety).

Some proteins may be recalcitrant to crystallization. However, severaltechniques are available to the skilled artisan to inducecrystallization. For example, the removal of flexible polypeptidesegments at the amino or carboxyl terminal end of the protein mayfacilitate production of crystalline protein samples. Removal of suchsegments can be done using molecular biology techniques or treatment ofthe protein with proteases such as trypsin, chymotrypsin, or subtilisin.

In diffraction experiments, a narrow and parallel beam of x-rays istaken from the x-ray source and directed onto the crystal to producediffracted beams. The incident primary beams cause damage to both themacromolecule and solvent molecules. The crystal is, therefore, cooled(e.g., to between −220° C. and −50° C.) to prolong its lifetime. Theprimary beam must strike the crystal from many directions to produce allpossible diffraction spots, so the crystal is rotated in the beam duringthe experiment. The diffracted spots are recorded on a film or by anelectronic detector. Exposed film has to be digitized and quantified ina scanning device, whereas the electronic detectors feed the signalsthey detect directly into a computer. Electronic area detectorssignificantly reduce the time required to collect and measurediffraction data. Each diffraction beam, which is recorded as a spot onfilm or a detector plate, is defined by three properties: the amplitude,which is measured from the intensity of the spot; the wavelength, whichis set by the x-ray source; and the phase, which is lost in x-rayexperiments. All three properties are needed for all of the diffractedbeams in order to determine the positions of the atoms giving rise tothe diffracted beams. One way of determining the phases is calledMultiple Isomorphous Replacement (MIR), which requires the introductionof exogenous x-ray scatterers (e.g., heavy atoms such metal atoms) intothe unit cell of the crystal. For a more detailed description of MIR,see U.S. Pat. No. 6,093,573 (column 15) the disclosure of which isincorporated herein by reference in its entirety.

Atomic coordinates refer to Cartesian coordinates (x, y, and zpositions) derived from mathematical equations involving Fouriersynthesis of data derived from patterns obtained via diffraction of amonochromatic beam of x-rays by the atoms (scattering centers) ofbiological macromolecule of interest in crystal form. Diffraction dataare used to calculate electron density maps of repeating units in thecrystal (unit cell). Electron density maps are used to establish thepositions (atomic coordinates) of individual atoms within a crystal'sunit cell. The absolute values of atomic coordinates convey spatialrelationships between atoms because the absolute values ascribed toatomic coordinates can be changed by rotational and/or translationalmovement along x, y, and/or z axes, together or separately, whilemaintaining the same relative spatial relationships among atoms. Thus, abiological macromolecule (e.g., a protein) whose set of absolute atomiccoordinate values can be rotationally or translationally adjusted tocoincide with a set of prior determined values from an analysis ofanother sample is considered to have the same atomic coordinates asthose obtained from the other sample.

Further details on x-ray crystallography can be obtained from co-pendingU.S. Application No. 2005/0015232, U.S. Pat. No. 6,093,573 andInternational Application Nos. PCT/US99/18441, PCT/US99/11913, andPCT/US00/03745. The disclosures of all these patent documents areincorporated herein by reference in their entirety.

NMR Spectroscopy.

Whereas x-ray crystallography requires single crystals of amacromolecule of interest, NMR measurements are carried out in solutionunder near physiological conditions. However, NMR-derived structures arenot as detailed as crystal-derived structures.

While the use of NMR spectroscopy was until relatively recently limitedto the elucidation of the 3-D structure of relatively small molecules(e.g., proteins of 100-150 amino acid residues), recent advancesincluding isotopic labeling of the molecule of interest and transverserelaxation-optimized spectroscopy (TROSY) have allowed the methodologyto be extended to the analysis of much larger molecules, e.g., proteinswith a molecular weight of 110 kDa (Wider, 2000).

NMR uses radio-frequency radiation to examine the environment ofmagnetic atomic nuclei in a homogeneous magnetic field pulsed with aspecific radio frequency. The pulses perturb the nuclear magnetizationof those atoms with nuclei of nonzero spin. Transient time domainsignals are detected as the system returns to equilibrium. Fouriertransformation of the transient signal into a frequency domain yields aone-dimensional NMR spectrum. Peaks in these spectra represent chemicalshifts of the various active nuclei. The chemical shift of an atom isdetermined by its local electronic environment. Two-dimensional NMRexperiments can provide information about the proximity of various atomsin the structure and in three dimensional space. Protein structures canbe determined by performing a number of two- (and sometimes 3- or 4-)dimensional NMR experiments and using the resulting information asconstraints in a series of protein folding simulations.

More information on NMR spectroscopy including detailed descriptions ofhow raw data obtained from an NMR experiment can be used to determinethe 3-D structure of a macromolecule can be found in: Protein NMRSpectroscopy, Principles and Practice, (1996); Gronenborn et al. (1990);and Wider (2000), supra., the disclosures of all of which areincorporated herein by reference in their entirety

Also of interest are peptidomimetic compounds that are designed basedupon the amino acid sequences of compounds of the invention that arepeptides. Peptidomimetic compounds are synthetic compounds having athree-dimensional conformation “motif” that is substantially the same asthe three-dimensional conformation of a selected peptide. The peptidemotif provides the peptidomimetic compound with the ability to inhibitthe interaction of α6β4 and HER2 or EGFR. Peptidomimetic compounds canhave additional characteristics that enhance their in vivo utility, suchas increased cell permeability and prolonged biological half-life. Thepeptidomimetics typically have a backbone that is partially orcompletely non-peptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

IV. METHODS OF TREATING HEART FAILURE

A. Therapeutic Regimens

Current medical management of cardiac hypertrophy in the setting of acardiovascular disorder includes the use of at least two types of drugs:inhibitors of the rennin-angiotensoin system, and β-adrenergic blockingagents (Bristow, 1999). Therapeutic agents to treat pathologichypertrophy in the setting of heart failure include angiotensin IIconverting enzyme (ACE) inhibitors and β-adrenergic receptor blockingagents (Eichhorn and Bristow, 1996). Other pharmaceutical agents thathave been disclosed for treatment of cardiac hypertrophy includeangiotensin II receptor antagonists (U.S. Pat. No. 5,604,251) andneuropeptide Y antagonists (WO 98/33791). Despite currently availablepharmaceutical compounds, prevention and treatment of cardiachypertrophy, and subsequent heart failure, continue to present atherapeutic challenge.

Non-pharmacological treatment is primarily used as an adjunct topharmacological treatment. One means of non-pharmacological treatmentinvolves reducing the sodium in the diet. In addition,non-pharmacological treatment also entails the elimination of certainprecipitating drugs, including negative inotropic agents (e.g., certaincalcium channel blockers and antiarrhythmic drugs like disopyramide),cardiotoxins (e.g., amphetamines), and plasma volume expanders (e.g.,nonsteroidal anti-inflammatory agents and glucocorticoids).

In one embodiment of the present invention, methods for the treatment ofheart failure utilizing inhibitors as described herein. For the purposesof the present application, treatment comprises reducing one or more ofthe symptoms of cardiac hypertrophy, such as reduced exercise capacity,reduced blood ejection volume, increased left ventricular end diastolicpressure, increased pulmonary capillary wedge pressure, reduced cardiacoutput, cardiac index, increased pulmonary artery pressures, increasedleft ventricular end systolic and diastolic dimensions, and increasedleft ventricular wall stress, wall tension and wall thickness-same forright ventricle. In addition, use of the disclosed inhibitors may delaydevelopment of year failure.

Treatment regimens would vary depending on the clinical situation.However, long term maintenance would appear to be appropriate in mostcircumstances. It also may be desirable treat hypertrophy with thedisclosed inhibitors intermittently, such as within brief window duringdisease progression.

B. Combined Therapy

In another embodiment, it is envisioned to use an inhibitor as describedherein combination with other therapeutic modalities. Thus, in additionto the therapies described above, one may also provide to the patientmore “standard” pharmaceutical cardiac therapies. Examples of othertherapies include, without limitation, so-called “beta blockers,”anti-hypertensives, cardiotonics, anti-thrombotics, vasodilators,hormone antagonists, iontropes, diuretics, endothelin antagonists,calcium channel blockers, phosphodiesterase inhibitors, ACE inhibitors,angiotensin type 2 antagonists and cytokine blockers/inhibitors, andHDAC inhibitors.

Combinations may be achieved by contacting cardiac cells with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the agent. Alternatively, the therapyusing the inhibitors of the present invention may precede or followadministration of the other agent(s) by intervals ranging from minutesto weeks. In embodiments where the other agent and expression constructare applied separately to the cell, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and expression construct would still beable to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one would typically contact the cellwith both modalities within about 12-24 hours of each other and, morepreferably, within about 6-12 hours of each other, with a delay time ofonly about 12 hours being most preferred. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of either aninhibitor according to the present invention, or the other agent will bedesired. In this regard, various combinations may be employed. By way ofillustration, where the inhibitor of according to the present inventionis “A” and the other agent is “B”, the following permutations based on 3and 4 total administrations are exemplary:

-   -   A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B        A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A        A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B        Other combinations are likewise contemplated.

C. Pharmacological Therapeutic Agents

Pharmacological therapeutic agents and methods of administration,dosages, etc., are well known to those of skill in the art (see forexample, the “Physicians Desk Reference,” Klaassen's “ThePharmacological Basis of Therapeutics,” “Remington's PharmaceuticalSciences,” and “The Merck Index, Eleventh Edition,” incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchinvidual determinations are within the skill of those of ordinary skillin the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present invention include an antihyperlipoproteinemic agent,an antiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, ablood coagulant, an antiarrhythmic agent, an antihypertensive agent, avasopressor, a treatment agent for congestive heart failure, anantianginal agent, an antibacterial agent or a combination thereof.

In addition, it should be noted that any of the following may be used todevelop new sets of cardiac therapy target genes as β-blockers were usedin the present examples (see below). While it is expected that many ofthese genes may overlap, new gene targets likely can be developed.

i. Antihyperlipoproteinemics

In certain embodiments, administration of an agent that lowers theconcentration of one of more blood lipids and/or lipoproteins, knownherein as an “antihyperlipoproteinemic,” may be combined with acardiovascular therapy according to the present invention, particularlyin treatment of athersclerosis and thickenings or blockages of vasculartissues. In certain aspects, an antihyperlipoproteinemic agent maycomprise an aryloxyalkanoic/fibric acid derivative, a resin/bile acidsequesterant, a HMG CoA reductase inhibitor, a nicotinic acidderivative, a thyroid hormone or thyroid hormone analog, a miscellaneousagent or a combination thereof

a. Aryloxyalkanoic Acid/Fibric Acid Derivatives

Non-limiting examples of aryloxyalkanoic/fibric acid derivatives includebeclobrate, enzafibrate, binifibrate, ciprofibrate, clinofibrate,clofibrate (atromide-S), clofibric acid, etofibrate, fenofibrate,gemfibrozil (lobid), nicofibrate, pirifibrate, ronifibrate, simfibrateand theofibrate.

b. Resins/Bile Acid Sequesterants

Non-limiting examples of resins/bile acid sequesterants includecholestyramine (cholybar, questran), colestipol (colestid) andpolidexide.

c. HMG CoA Reductase Inhibitors

Non-limiting examples of HMG CoA reductase inhibitors include lovastatin(mevacor), pravastatin (pravochol) or simvastatin (zocor).

d. Nicotinic Acid Derivatives

Non-limiting examples of nicotinic acid derivatives include nicotinate,acepimox, niceritrol, nicoclonate, nicomol and oxiniacic acid.

e. Thyroid Hormones and Analogs

Non-limiting examples of thyroid hormones and analogs thereof includeetoroxate, thyropropic acid and thyroxine.

f. Miscellaneous Antihyperlipoproteinemics

Non-limiting examples of miscellaneous antihyperlipoproteinemics includeacifran, azacosterol, benfluorex, β-benzalbutyramide, carnitine,chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium,5,8,11,14,17-eicosapentaenoic acid, eritadenine, furazabol, meglutol,melinamide, mytatrienediol, ornithine, γ-oryzanol, pantethine,pentaerythritol tetraacetate, α-phenylbutyramide, pirozadil, probucol(lorelco), β-sitosterol, sultosilic acid-piperazine salt, tiadenol,triparanol and xenbucin.

ii. Antiarteriosclerotics

Non-limiting examples of an antiarteriosclerotic include pyridinolcarbamate.

iii. Antithrombotic/Fibrinolytic Agents

In certain embodiments, administration of an agent that aids in theremoval or prevention of blood clots may be combined with administrationof a modulator, particularly in treatment of athersclerosis andvasculature (e.g., arterial) blockages. Non-limiting examples ofantithrombotic and/or fibrinolytic agents include anticoagulants,anticoagulant antagonists, antiplatelet agents, thrombolytic agents,thrombolytic agent antagonists or combinations thereof

In certain aspects, antithrombotic agents that can be administeredorally, such as, for example, aspirin and wafarin (coumadin), arepreferred.

a. Anticoagulants

A non-limiting example of an anticoagulant include acenocoumarol,ancrod, anisindione, bromindione, clorindione, coumetarol, cyclocumarol,dextran sulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate,ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodium,oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin,picotamide, tioclomarol and warfarin.

b. Antiplatelet Agents

Non-limiting examples of antiplatelet agents include aspirin, a dextran,dipyridamole (persantin), heparin, sulfinpyranone (anturane) andticlopidine (ticlid).

c. Thrombolytic Agents

Non-limiting examples of thrombolytic agents include tissue plaminogenactivator (activase), plasmin, pro-urokinase, urokinase (abbokinase)streptokinase (streptase), anistreplase/APSAC (eminase).

iv. Blood Coagulants

In certain embodiments wherein a patient is suffering from a hemmorageor an increased likelyhood of hemmoraging, an agent that may enhanceblood coagulation may be used. Non-limiting examples of a bloodcoagulation promoting agent include thrombolytic agent antagonists andanticoagulant antagonists.

a. Anticoagulant Antagonists

Non-limiting examples of anticoagulant antagonists include protamine andvitamine K1.

b. Thrombolytic Agent Antagonists and Antithrombotics

Non-limiting examples of thrombolytic agent antagonists includeamiocaproic acid (amicar) and tranexamic acid (amstat). Non-limitingexamples of antithrombotics include anagrelide, argatroban, cilstazol,daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan,ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.

v. Antiarrhythmic Agents

Non-limiting examples of antiarrhythmic agents include Class Iantiarrythmic agents (sodium channel blockers), Class II antiarrythmicagents (beta-adrenergic blockers), Class II antiarrythmic agents(repolarization prolonging drugs), Class IV antiarrhythmic agents(calcium channel blockers) and miscellaneous antiarrythmic agents.

a. Sodium Channel Blockers

Non-limiting examples of sodium channel blockers include Class IA, ClassIB and Class IC antiarrhythmic agents. Non-limiting examples of Class IAantiarrhythmic agents include disppyramide (norpace), procainamide(pronestyl) and quinidine (quinidex). Non-limiting examples of Class IBantiarrhythmic agents include lidocaine (xylocalne), tocamide (tonocard)and mexiletine (mexitil). Non-limiting examples of Class ICantiarrhythmic agents include encamide (enkaid) and flecamide(tambocor).

b. Beta Blockers

Non-limiting examples of a beta blocker, otherwise known as aβ-adrenergic blocker, a β-adrenergic antagonist or a Class IIantiarrhythmic agent, include acebutolol (sectral), alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol,bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol (brevibloc), indenolol, labetalol, levobunolol, mepindolol,metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol,nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol,propanolol (inderal), sotalol (betapace), sulfinalol, talinolol,tertatolol, timolol, toliprolol and xibinolol. In certain aspects, thebeta blocker comprises an aryloxypropanolamine derivative. Non-limitingexamples of aryloxypropanolamine derivatives include acebutolol,alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol,bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol,celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol,metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol,pindolol, propanolol, talinolol, tertatolol, timolol and toliprolol.

c. Repolarization Prolonging Agents

Non-limiting examples of an agent that prolong repolarization, alsoknown as a Class III antiarrhythmic agent, include amiodarone(cordarone) and sotalol (betapace).

d. Calcium Channel Blockers/Antagonist

Non-limiting examples of a calcium channel blocker, otherwise known as aClass IV antiarrythmic agent, include an arylalkylamine (e.g.,bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline,verapamil), a dihydropyridine derivative (felodipine, isradipine,nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) apiperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) ora micellaneous calcium channel blocker such as bencyclane, etafenone,magnesium, mibefradil or perhexyline. In certain embodiments a calciumchannel blocker comprises a long-acting dihydropyridine(nifedipine-type) calcium antagonist.

e. Miscellaneous Antiarrhythmic Agents

Non-limiting examples of miscellaneous antiarrhymic agents includeadenosine (adenocard), digoxin (lanoxin), acecamide, ajmaline,amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine,capobenic acid, cifenline, disopyranide, hydroquinidine, indecamide,ipatropium bromide, lidocaine, lorajmine, lorcamide, meobentine,moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidinepolygalacturonate, quinidine sulfate and viquidil.

vi. Antihypertensive Agents

Non-limiting examples of antihypertensive agents include sympatholytic,alpha/beta blockers, alpha blockers, anti-angiotensin II agents, betablockers, calcium channel blockers, vasodilators and miscellaneousantihypertensives.

a. Alpha Blockers

Non-limiting examples of an alpha blocker, also known as an α-adrenergicblocker or an α-adrenergic antagonist, include amosulalol, arotinolol,dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin,labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin andyohimbine. In certain embodiments, an alpha blocker may comprise aquinazoline derivative. Non-limiting examples of quinazoline derivativesinclude alfuzosin, bunazosin, doxazosin, prazosin, terazosin andtrimazosin.

b. Alpha/Beta Blockers

In certain embodiments, an antihypertensive agent is both an alpha andbeta adrenergic antagonist. Non-limiting examples of an alpha/betablocker comprise labetalol (normodyne, trandate).

c. Anti-Angiotension II Agents

Non-limiting examples of anti-angiotension II agents include angiotensinconverting enzyme inhibitors and angiotension II receptor antagonists.Non-limiting examples of angiotension converting enzyme inhibitors (ACEinhibitors) include alacepril, enalapril (vasotec), captopril,cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril,perindopril, quinapril and ramipril. Non-limiting examples of anangiotensin II receptor blocker, also known as an angiotension IIreceptor antagonist, an ANG receptor blocker or an ANG-II type-1receptor blocker (ARBS), include angiocandesartan, eprosartan,irbesartan, losartan and valsartan.

d. Sympatholytics

Non-limiting examples of a sympatholytic include a centrally actingsympatholytic or a peripherially acting sympatholytic. Non-limitingexamples of a centrally acting sympatholytic, also known as an centralnervous system (CNS) sympatholytic, include clonidine (catapres),guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet).Non-limiting examples of a peripherally acting sympatholytic include aganglion blocking agent, an adrenergic neuron blocking agent, aβ-adrenergic blocking agent or a alpha1-adrenergic blocking agent.Non-limiting examples of a ganglion blocking agent include mecamylamine(inversine) and trimethaphan (arfonad). Non-limiting of an adrenergicneuron blocking agent include guanethidine (ismelin) and reserpine(serpasil). Non-limiting examples of a β-adrenergic blocker includeacenitolol (sectral), atenolol (tenormin), betaxolol (kerlone),carteolol (cartrol), labetalol (normodyne, trandate), metoprolol(lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken),propranolol (inderal) and timolol (blocadren). Non-limiting examples ofalpha1-adrenergic blocker include prazosin (minipress), doxazocin(cardura) and terazosin (hytrin).

e. Vasodilators

In certain embodiments a cardiovasculator therapeutic agent may comprisea vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or aperipheral vasodilator). In certain preferred embodiments, a vasodilatorcomprises a coronary vasodilator. Non-limiting examples of a coronaryvasodilator include amotriphene, bendazol, benfurodil hemisuccinate,benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep,dipyridamole, droprenilamine, efloxate, erythrityl tetranitrane,etafenone, fendiline, floredil, ganglefene, herestrolbis(β-diethylaminoethyl ether), hexobendine, itramin tosylate, khellin,lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin,pentaerythritol tetranitrate, pentrinitrol, perhexyline, pimethylline,trapidil, tricromyl, trimetazidine, troInitrate phosphate and visnadine.

In certain aspects, a vasodilator may comprise a chronic therapyvasodilator or a hypertensive emergency vasodilator. Non-limitingexamples of a chronic therapy vasodilator include hydralazine(apresoline) and minoxidil (loniten). Non-limiting examples of ahypertensive emergency vasodilator include nitroprusside (nipride),diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten)and verapamil.

f. Miscellaneous Antihypertensives

Non-limiting examples of miscellaneous antihypertensives includeajmaline, γ-aminobutyric acid, bufeniode, cicletainine, ciclosidomine, acryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate,mecamylamine, methyldopa, methyl 4-pyridyl ketone thiosemicarbazone,muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, aprotoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodiumnitrorusside, ticrynafen, trimethaphan camsylate, tyrosinase andurapidil.

In certain aspects, an antihypertensive may comprise an arylethanolaminederivative, a benzothiadiazine derivative, aN-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative,a guanidine derivative, a hydrazines/phthalazine, an imidazolederivative, a quanternary ammonium compound, a reserpine derivative or asuflonamide derivative.

Arylethanolamine Derivatives. Non-limiting examples of arylethanolaminederivatives include amosulalol, bufuralol, dilevalol, labetalol,pronethalol, sotalol and sulfinalol.

Benzothiadiazine Derivatives. Non-limiting examples of benzothiadiazinederivatives include althizide, bendroflumethiazide, benzthiazide,benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone,cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide,fenquizone, hydrochlorothizide, hydroflumethizide, methyclothiazide,meticrane, metolazone, paraflutizide, polythizide, tetrachlormethiazideand trichlormethiazide.

N-carboxyalkyl(peptide/lactam) Derivatives. Non-limiting examples ofN-carboxyalkyl(peptide/lactam) derivatives include alacepril, captopril,cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril,moveltipril, perindopril, quinapril and ramipril.

Dihydropyridine Derivatives. Non-limiting examples of dihydropyridinederivatives include amlodipine, felodipine, isradipine, nicardipine,nifedipine, nilvadipine, nisoldipine and nitrendipine.

Guanidine Derivatives. Non-limiting examples of guanidine derivativesinclude bethanidine, debrisoquin, guanabenz, guanacline, guanadrel,guanazodine, guanethidine, guanfacine, guanochlor, guanoxabenz andguanoxan.

Hydrazines/Phthalazines. Non-limiting examples ofhydrazines/phthalazines include budralazine, cadralazine, dihydralazine,endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine andtodralazine.

Imidazole Derivatives. Non-limiting examples of imidazole derivativesinclude clonidine, lofexidine, phentolamine, tiamenidine and tolonidine.

Quanternary Ammonium Compounds. Non-limiting examples of quanternaryammonium compounds include azamethonium bromide, chlorisondaminechloride, hexamethonium, pentacynium bis(methylsulfate), pentamethoniumbromide, pentolinium tartrate, phenactropinium chloride andtrimethidinium methosulfate.

Reserpine Derivatives. Non-limiting examples of reserpine derivativesinclude bietaserpine, deserpidine, rescinnamine, reserpine andsyrosingopine.

Suflonamide Derivatives. Non-limiting examples of sulfonamidederivatives include ambuside, clopamide, furosemide, indapamide,quinethazone, tripamide and xipamide.

g. Vasopressors

Vasopressors generally are used to increase blood pressure during shock,which may occur during a surgical procedure. Non-limiting examples of avasopressor, also known as an antihypotensive, include amezinium methylsulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin,gepefrine, metaraminol, midodrine, norepinephrine, pholedrine andSynephrine.

vii. Treatment Agents for Congestive Heart Failure

Non-limiting examples of agents for the treatment of congestive heartfailure include anti-angiotension II agents, afterload-preload reductiontreatment, diuretics and inotropic agents.

a. Afterload-Preload Reduction

In certain embodiments, an animal patient that can not tolerate anangiotension antagonist may be treated with a combination therapy. Suchtherapy may combine administration of hydralazine (apresoline) andisosorbide dinitrate (isordil, sorbitrate).

b. Diuretics

Non-limiting examples of a diuretic include a thiazide orbenzothiadiazine derivative (e.g., althiazide, bendroflumethazide,benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide,chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide,ethiazide, ethiazide, fenquizone, hydrochlorothiazide,hydroflumethiazide, methyclothiazide, meticrane, metolazone,paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide),an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide,mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurouschloride, mersalyl), a pteridine (e.g., furtherene, triamterene),purines (e.g., acefylline, 7-morpholinomethyltheophylline, pamobrom,protheobromine, theobromine), steroids including aldosterone antagonists(e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative(e.g., acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone,diphenylmethane-4,4′-disulfonamide, disulfamide, ethoxzolamide,furosemide, indapamide, mefruside, methazolamide, piretanide,quinethazone, torasemide, tripamide, xipamide), a uracil (e.g.,aminometradine, amisometradine), a potassium sparing antagonist (e.g.,amiloride, triamterene) or a miscellaneous diuretic such as aminozine,arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine,isosorbide, mannitol, metochalcone, muzolimine, perhexyline, ticrnafenand urea.

c. Inotropic Agents

Non-limiting examples of a positive inotropic agent, also known as acardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline,amrinone, benfurodil hemisuccinate, bucladesine, cerberosine,camphotamide, convallatoxin, cymarin, denopamine, deslanoside,digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine,dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin,glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside,metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine,prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin,strphanthin, sulmazole, theobromine and xamoterol.

In particular aspects, an intropic agent is a cardiac glycoside, abeta-adrenergic agonist or a phosphodiesterase inhibitor. Non-limitingexamples of a cardiac glycoside includes digoxin (lanoxin) and digitoxin(crystodigin). Non-limiting examples of a β-adrenergic agonist includealbuterol, bambuterol, bitolterol, carbuterol, clenbuterol,clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex),dopamine (intropin), dopexamine, ephedrine, etafedrine,ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine,isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine,oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol,ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol andxamoterol. Non-limiting examples of a phosphodiesterase inhibitorinclude aminone (inocor).

d. Antianginal Agents

Antianginal agents may comprise organonitrates, calcium channelblockers, beta blockers and combinations thereof.

Non-limiting examples of organonitrates, also known asnitrovasodilators, include nitroglycerin (nitro-bid, nitrostat),isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol,vaporole).

D. Surgical Therapeutic Agents

In certain aspects, the secondary therapeutic agent may comprise asurgery of some type, which includes, for example, preventative,diagnostic or staging, curative and palliative surgery. Surgery, and inparticular a curative surgery, may be used in conjunction with othertherapies, such as the present invention and one or more other agents.

Such surgical therapeutic agents for vascular and cardiovasculardiseases and disorders are well known to those of skill in the art, andmay comprise, but are not limited to, performing surgery on an organism,providing a cardiovascular mechanical prostheses, angioplasty, coronaryartery reperfusion, catheter ablation, providing an implantablecardioverter defibrillator to the subject, mechanical circulatorysupport or a combination thereof. Non-limiting examples of a mechanicalcirculatory support that may be used in the present invention comprisean intra-aortic balloon counterpulsation, left ventricular assist deviceor combination thereof.

E. Drug Formulations and Routes for Administration to Patients

Where clinical applications are contemplated, pharmaceuticalcompositions will be prepared in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector or cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes solvents,buffers, solutions, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the likeacceptable for use in formulating pharmaceuticals, such aspharmaceuticals suitable for administration to humans. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients of the present invention, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions, providedthey do not inactivate the vectors or cells of the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route. This includes oral,nasal, or buccal. Alternatively, administration may be by intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection,or by direct injection into cardiac tissue. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions, asdescribed supra.

The active compounds may also be administered parenterally orintraperitoneally. By way of illustration, solutions of the activecompounds as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. Generally, these preparations are sterile and fluid to theextent that easy injectability exists. Preparations should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. Appropriate solvents or dispersion media may contain, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the desired otheringredients, e.g., as enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation include vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient(s) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

For oral administration the polypeptides of the present inventiongenerally may be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

The compositions of the present invention generally may be formulated ina neutral or salt form. Pharmaceutically-acceptable salts include, forexample, acid addition salts (formed with the free amino groups of theprotein) derived from inorganic acids (e.g., hydrochloric or phosphoricacids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups of the proteincan also be derived from inorganic bases (e.g., sodium, potassium,ammonium, calcium, or ferric hydroxides) or from organic bases (e.g.,isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety, and purity standards as required by FDA Office ofBiologics standards.

V. PURIFICATION OF PEPTIDES/PROTEINS

It will be desirable to purify peptides and polypeptides according tothe present invention. Protein purification techniques are well known tothose of skill in the art. These techniques involve, at one level, thecrude fractionation of the cellular milieu to polypeptide andnon-polypeptide fractions. Having separated the polypeptide from otherproteins, the polypeptide of interest may be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

VI. DEFINITIONS

As used herein, the term “heart failure” is broadly used to mean anycondition that reduces the ability of the heart to pump blood. As aresult, congestion and edema develop in the tissues. Most frequently,heart failure is caused by decreased contractility of the myocardium,resulting from reduced coronary blood flow; however, many other factorsmay result in heart failure, including damage to the heart valves,vitamin deficiency, and primary cardiac muscle disease. Though theprecise physiological mechanisms of heart failure are not entirelyunderstood, heart failure is generally believed to involve disorders inseveral cardiac autonomic properties, including sympathetic,parasympathetic, and baroreceptor responses. The phrase “manifestationsof heart failure” is used broadly to encompass all of the sequelaeassociated with heart failure, such as shortness of breath, pittingedema, an enlarged tender liver, engorged neck veins, pulmonary ralesand the like including laboratory findings associated with heartfailure.

The term “treatment” or grammatical equivalents encompasses theimprovement and/or reversal of the symptoms of heart failure (i.e., theability of the heart to pump blood). “Improvement in the physiologicfunction” of the heart may be assessed using any of the measurementsdescribed herein (e.g., measurement of ejection fraction, fractionalshortening, left ventricular internal dimension, heart rate, etc.), aswell as any effect upon the animal's survival. In use of animal models,the response of treated transgenic animals and untreated transgenicanimals is compared using any of the assays described herein (inaddition, treated and untreated non-transgenic animals may be includedas controls). A compound which causes an improvement in any parameterassociated with heart failure used in the screening methods of theinstant invention may thereby be identified as a therapeutic compound.

The term “dilated cardiomyopathy” refers to a type of heart failurecharacterized by the presence of a symmetrically dilated left ventriclewith poor systolic contractile function and, in addition, frequentlyinvolves the right ventricle.

The term “compound” refers to any chemical entity, pharmaceutical, drug,and the like that can be used to treat or prevent a disease, illness,sickness, or disorder of bodily function. Compounds comprise both knownand potential therapeutic compounds. A compound can be determined to betherapeutic by screening using the screening methods of the presentinvention. A “known therapeutic compound” refers to a therapeuticcompound that has been shown (e.g., through animal trials or priorexperience with administration to humans) to be effective in suchtreatment. In other words, a known therapeutic compound is not limitedto a compound efficacious in the treatment of heart failure.

As used herein, the term “cardiac hypertrophy” refers to the process inwhich adult cardiac myocytes respond to stress through hypertrophicgrowth. Such growth is characterized by cell size increases without celldivision, assembling of additional sarcomeres within the cell tomaximize force generation, and an activation of a fetal cardiac geneprogram. Cardiac hypertrophy is often associated with increased risk ofmorbidity and mortality, and thus studies aimed at understanding themolecular mechanisms of cardiac hypertrophy could have a significantimpact on human health.

As used herein, the terms “antagonist” and “inhibitor” refer tomolecules, compounds, or nucleic acids which inhibit the action of acellular factor that may be involved in cardiac hypertrophy. Antagonistsmay or may not be homologous to these natural compounds in respect toconformation, charge or other characteristics. Thus, antagonists may berecognized by the same or different receptors that are recognized by anagonist. Antagonists may have allosteric effects which prevent theaction of an agonist. Alternatively, antagonists may prevent thefunction of the agonist. In contrast to the agonists, antagonisticcompounds do not result in pathologic and/or biochemical changes withinthe cell such that the cell reacts to the presence of the antagonist inthe same manner as if the cellular factor was present. Antagonists andinhibitors may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind or interact with a receptor, molecule, and/orpathway of interest.

As used herein, the term “modulate” refers to a change or an alterationin a biological activity. Modulation may be an increase or a decrease inprotein activity, a change in kinase activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties associated with the activity of a protein orother structure of interest. The term “modulator” refers to any moleculeor compound which is capable of changing or altering biological activityas described above.

The term “β-adrenergic receptor antagonist” refers to a chemicalcompound or entity that is capable of blocking, either partially orcompletely, the beta (β) type of adrenoreceptors (i.e., receptors of theadrenergic system that respond to catecholamines, especiallynorepinephrine). Some β-adrenergic receptor antagonists exhibit a degreeof specificity for one receptor sybtype (generally β₁); such antagonistsare termed “β₁-specific adrenergic receptor antagonists” and“β₂-specific adrenergic receptor antagonists.” The term β-adrenergicreceptor antagonist” refers to chemical compounds that are selective andnon-selective antagonists. Examples of β-adrenergic receptor antagonistsinclude, but are not limited to, acebutolol, atenolol, butoxamine,carteolol, esmolol, labetolol, metoprolol, nadolol, penbutolol,propanolol, and timolol. The use of derivatives of known β-adrenergicreceptor antagonists is encompassed by the methods of the presentinvention. Indeed any compound, which functionally behaves as aβ-adrenergic receptor antagonist is encompassed by the methods of thepresent invention.

The terms “angiotensin-converting enzyme inhibitor” or “ACE inhibitor”refer to a chemical compound or entity that is capable of inhibiting,either partially or completely, the enzyme involved in the conversion ofthe relatively inactive angiotensin I to the active angiotensin II inthe rennin-angiotensin system. In addition, the ACE inhibitorsconcomitantly inhibit the degradation of bradykinin, which likelysignificantly enhances the antihypertensive effect of the ACEinhibitors. Examples of ACE inhibitors include, but are not limited to,benazepril, captopril, enalopril, fosinopril, lisinopril, quiapril andramipril. The use of derivatives of known ACE inhibitors is encompassedby the methods of the present invention. Indeed any compound, whichfunctionally behaves as an ACE inhibitor, is encompassed by the methodsof the present invention.

VII. EXAMPLES

The following examples are included to further illustrate variousaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques and/or compositions discovered by the inventor tofunction well in the practice of the invention, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Example 1 Materials and Methods

Development of the Peptide Disruptor of cMyBP-C Binding to Myosin.

A myosin-binding peptide-based therapeutic agent has been identifiedbased on the premise that disruption of the myosin-cMyBP-C interfacewould release a molecular brake on cardiomyocyte contractility imposedby the inhibitory activity of the unphosphorylated form of cMyBP-C onmyosin. Since there is no PDB-reported structure for the 11-domainprotein cMyBP-C, the molecular design of the peptide was developed on asequence-based approximate prediction of a putative myosin-binding site.To predict the sequence of the peptide, the inventors sought a regionbetween the C1 and C2 domains of cMyBP-C (which binds to myosin) in thetwilight zone between order and disorder, using PONDR®, a predictor ofnative disorder (Pietrosemoli et al., 2007). The version used,PONDR-VLXT (Li et al., 1999), assigns a disorder propensity score D toeach amino acid along the chain, with D=0 corresponding to certainty oforder and D=1 corresponding to certainty of structural disorder. On theother hand, it is well established that regions in the twilight betweenorder and disorder, with 0.35<D<0.8, are rich in structural defectsknown as “dehydrons,” which signal local sites in the structure withsolvent exposure of backbone hydrogen bonds. These regions areinherently sticky since dehydrons enhance their stability by promotingdehydration (Fernandez, 2012), which in turn translates mechanicallyinto an attractive drag on nonpolar groups.

A crucial twilight region containing phosphorylation sites S302, S307was identified in the motif region intercalated between domains C1 andC2 of cMyBP-C, more precisely in the sequence region 293-310. Thus, the18-unit region ₂₉₃FSSLLKKRDSFRRDSKLE₃₁₀ (SEQ ID NO:46) was predicted asdisordered in the 302,307-phosphorylated state but capable of atransition to an ordered state upon binding to myosin in theunphosphorylated state. This finding guided the molecular design of thepeptide 302A: FSSLLKKRDAFRRDAKLE (SEQ ID NO:47), with S→A substitutionsat positions 302 and 307. Peptide 302A becomes a surrogate for amyosin-binding region of cMyBP-C, susceptible to acquiring order uponbinding to myosin, while incapable of being phosphorylated and thusreversing back to the unbound state. These properties provide therationale for its therapeutic impact to treat heart failure, based onits competitive binding to myosin and concurrent release of themolecular brake on contractility by precluding binding of cMyBP-C.

The peptide was synthesized by a commercial vendor (UW BiotechnologyCenter) and delivered as a lyophilized powder. The peptide was dissolvedin water and subsequently dialyzed against water to remove ioniccontaminants, lyophilized and then dissolved in relaxing solution(below) for treatment of membrane permeabilized (skinned) preparationsof murine and porcine myocardium.

Contractile measurements. The experimental methods used in testing thepeptide disruptor of cMyBP-C binding to myosin are standard methods inthe inventors' laboratory, as published previously (e.g., Chen et al.,2010).

Skinned myocardial preparations. Skinned ventricular myocardium wasprepared according to the protocol described by Chen et al. (2010). Inbrief, beating hearts were excised in vivo from anesthetized WT mice ofeither sex (˜6 months old) and dissected in Ca²⁺-Ringer's solutioncontaining 120 mM NaCl, 19 mM NaHCO₃, 5 mM KCl, 1.2 mM Na₂HPO₄, 1.2 mMMgSO₄, 1 mM CaCl₂, and 10 mM glucose, pH 7.4 at 22° C., pre-equilibratedwith 95% O₂/5% CO₂. Basal levels of RLC phosphorylation were reduced touniformly low levels (i.e., <10%) as described previously (Olsson etal., 2004; Stelzer et al., 2006) by perfusing hemisected hearts withCa²⁺-Ringer's solution containing 30 mM 2,3-butanedione monoxime (BDM)for 30 minutes at 22° C. before rapid freezing in liquid nitrogen forstorage prior to experimental measurements. The dephosphorylation of RLCprovides an appropriate background for assessment of the effects of thepeptide, since RLC phosphorylation increases force production in heartmuscle and speeds contraction and RLC phosphorylation is nearly nil inheart failure.

On the day of an experiment, frozen ventricles were thawed andhomogenized in ice-cold relaxing solution containing 100 mM KCl, 10 mMimidazole, 5 mM MgCl₂, 2 mM EGTA, and 4 mMATP, pH 7.0 at 22° C. to yieldmulticellular preparations with dimensions of 600-900 μm×100-250 μmusing a Polytron homogenizer. The cellular homogenates were thencentrifuged and subsequently resuspended in ice-cold relaxing solutioncontaining 250 μg/ml saponin and 1% Triton X-100 for 30 minutes at 4° C.The cellular homogenates were centrifuged, washed with ice-cold relaxingsolution several times and stored on ice until used in mechanicalexperiments.

Measurements of Force and the Kinetics of Force Development.

Skinned myocardial preparations were attached between a force transducer(for measurements of force) and a displacement motor (to impose rapidchanges in length for measurements of the kinetics of force development)while in relaxing solution (Olsson et al., 2004). The temperature wasthen adjusted to 22° C. for measurements of the contractile propertiesof myocardium in the presence and absence of peptide.

The force generating properties of murine or porcine skinned myocardiumwere measured as a function of free [Ca²⁺] in activating solutionscontaining salts, a Ca²⁺ buffer, and MgATP (Olsson et al., 2004). FreeCa²⁺, expressed as −log [Ca²⁺]_(free), was varied between sub-threshold(pCa 7.0) and saturating (pCa 4.5) levels with respect to forcedevelopment. Measurements of isometric force as a function of pCayielded force-pCa relationships, from which was determined the Ca²⁺sensitivity of force defined as the pCa at which force was half-maximal.

The kinetics of force development were assessed as a function of pCa bymeasuring the rate of force redevelopment at each pCa following a rapidslackening and re-stretch of the muscle during steady Ca²⁺ activation(Olsson et al., 2004; Stelzer et al., 2006). Fitting an exponentialcurve to the time-course of force redevelopment following this sequenceof length changes yielded a rate constant of force redevelopment(k_(tr)). Kinetic data are presented as plots of k_(tr) vs the isometricforce developed at each pCa.

The force and kinetic data presented in the following section wereobtained from each muscle preparation first during maximal activation atpCa 4.5, next during submaximal activation at a pCa>4.5, and once againduring activation at pCa 4.5. The preparation was then bathed inrelaxing solution containing peptide, after which the identicalactivation sequence was imposed. Finally, the peptide was washed out ofthe preparation by repeated washings in relaxing solution so thatcontrol measurements could again be done in the absence of peptide. Thisbracketing protocol ensured that the results in the presence of peptidewere corrected for time-dependent rundown of the preparations during theexperiment.

Porcine Myocardium.

Measurements of Ca²⁺ sensitivity of force were also performed usingskinned myocardium from pig hearts, since these hearts are more similarto human hearts with respect to protein isoform expression than aremouse hearts. Specifically, the principal myosin isoform in porcine andhuman hearts is the much slower β-cardiac myosin, while the principalisoform in mouse hearts is the faster α-cardiac myosin. The developmentof force in cardiac muscle exhibits positive cooperativity in thatbinding of myosin to actin enhances the activation state of the thinfilament, which recruits additional myosins to bind thereby increasingforce (Moss and Fitzsimons, 2010). The slower β-cardiac isoform would bepredicted to have a greater activating effect on the cardiac thinfilament due to the longer residency time of the slower myosin on thethin filament.

Example 2 Results

Murine Myocardium.

The peptide disruptor of MyBP-C binding to myosin (called peptide“302a”) was infused into mouse skinned myocardium at a concentration of50 μM (FIG. 1). Force at each pCa was increased by the peptide,resulting in a shift of the force-pCa relationship to lower Ca²⁺concentrations, i.e., the pCa₅₀ increased from xxx in the control to xxxfollowing peptide treatment. Treatment of the preparations with ascrambled peptide had no effect on the force-pCa relationship,indicating that the peptide is a specific activator of myocardial force.

Peptide 302a also accelerated the rate of force development at each pCastudied in that the ktr-pCa relationship was shifted to lower Ca²⁺concentrations when the preparation was treated with 50 μM 302a, but notwhen treated with scrambled peptide (FIG. 2). Consistent with thisprediction, the effects of the 302a peptide on force development weresignificantly greater in porcine than in murine skinned myocardium (FIG.3).

FIG. 4 shows various particular peptides used in studies shown in FIGS.5-7. FIG. 5 show that scrambled or phosphomimetic (302A→D) 18-merpeptides have no activity at 50 μM on the force-pCa relationship. Thescrambled peptide was tested on mouse myocardium using a sequencegenerated randomly from the 302A base peptide. The 302D peptide wastested on porcine myocardium and is a phosphomimetic that was predictedto have no effect on the force-pCa relationship, since phosphorylationof native MyBP-C in situ disrupts its interaction with myosin in vitroand in vivo. Thus, no effect is the predicted result.

FIG. 6 shows the relative effects of various peptides on force at pCa5.9 (scaled to force at pCa 4.5) in mouse. At this high pCa (low[Ca²⁺]), force in untreated skinned myocardium is ˜8% of maximum forceat pCa 4.5. At 50 μM, peptide 302A increases force to nearly 3×. At 50μM, peptide 302A-[−2]C causes a further increase in force, while302A-[−4]_(C) mitigates this increase, i.e., has an effect approximatelyequal to the 302A base peptide. All peptides increased force relative tocontrol but the increases were smaller than those observed with 302A.Thus, the 302A-[4]_(C) (16-mer) peptide has greater effects than the302A peptide, while the 302A-[−4]_(C) (14-mer) peptide has effectssimilar to the 302A peptide. N-terminal-truncated peptides increaseforce but these increases are smaller than observed with the 302Apeptide.

FIG. 7 showns the relative effects of various peptides on force at pCa5.9 (scaled to force at pCa 4.5) in mouse. At this intermediate pCa,force in untreated myocardium is ˜35% of maximum force at pCa 4.5. Thedata show that at 50 μM, peptide 302A increases force to nearly 2×control. Furthermore, truncated peptides increased force by amounts thatwere similar to 302A, i.e., 302A-[−2]_(C), slightly less than 302A(302A-[−4]_(C), 302A-[−4]_(N)) or less than 302A (302A-[−4]_(N),302A-[−8]). Thus, all_truncated 302A peptides increased forcedevelopment, but only the 302A-[−2]_(C) (16-mer) increased force to thesame extent as the full-length 302A peptide.

CONCLUSIONS

From these results, the inventors conclude that a peptide disruptor ofthe binding of MyBP-C to cardiac muscle increases the force developed ateach concentration of free Ca²⁺ tested and also increased the rate offorce development. Since living myocardium operates at an intracellularCa²⁺ concentration that would elicit forces that are approximatelyhalf-maximal, the effects observed here in skinned myocardium predictthat the peptide would nearly double the force and rate of forcedevelopment in vivo. Also, because the force developed by myocardium isdiminished in heart failure, i.e., less than half-maximal, the peptidehas the potential to increase developed force several fold in failingmyocardium.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods, and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

VIII. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 5,440,013-   U.S. Pat. No. 5,446,128-   U.S. Pat. No. 5,475,085-   U.S. Pat. No. 5,597,457-   U.S. Pat. No. 5,604,251-   U.S. Pat. No. 5,618,914-   U.S. Pat. No. 5,670,155-   U.S. Pat. No. 5,672,681-   U.S. Pat. No. 5,674,976-   U.S. Pat. No. 5,710,245-   U.S. Pat. No. 5,790,421-   U.S. Pat. No. 5,840,833-   U.S. Pat. No. 5,859,184-   U.S. Pat. No. 5,889,155-   U.S. Pat. No. 5,929,237-   U.S. Pat. No. 6,093,573-   U.S. Pat. No. 6,261,569-   U.S. Pat. No. 7,183,059-   U.S. Pat. No. 7,192,713-   U.S. Publication Application No. 2005/0015232-   Bodanszky et al., J. Antibiot., 29(5):549-53, 1976.-   Bristow, Cardiology, 92:3-6, 1999.-   Capaldi et al., Biochem. Biophys. Res. Comm., 74(2):425-433, 1977.-   Chen et al., J. Gen. Physiol., 136:615-627, 2010.-   Cohen et al., J. Med. Chem., 33:883-894, 1990.-   Durand et al., Ann. Med., 27:311-317, 1995.-   Eichhorn and Bristow, Circulation, 94:2285-2296, 1996.-   Fernández, Phys. Rev. Lett., 108:188102, 2012.-   Fischer, Med. Res. Rev., 27(6):755-796, 2007.-   Gronenborn et al., Anal. Chem., 62(1):2-15, 1990.-   Jackson, Seminars in Oncology, 24:L164-172, 1997.-   Johnson et al., In: Biotechnology and Pharmacy, Pezzuto et al.,    eds., Chapman and Hall, New York, 1993.-   Jones et al., J. Med. Chem., 39:904-917, 1996.-   Klaassen, In: The Pharmacological Basis of Therapeutics, Goodman and    Gilman, Eds., Pergamon Press, 8^(th) Ed., 1990.-   Li et al., Gen. Inf., 10:30-40, 1999.-   McPherson, J. Biol. Chem., 251:6300-6306, 1976.-   Merrifield, J. Am. Chem. Soc., 85(14):2149-2154, 1963.-   Moss and Fitzsmons, J. Gen. Physiol., 136:21-27, 2010.-   Navia et al., Curr. Opin. Struct. Biol., 2:202-210, 1992.-   Olsson et al., Am. J. Physiol., 287:H2712-H2718, 2004.-   PCT Appln. PCT/US00/03745-   PCT Appln. PCT/US00/14667-   PCT Appln. PCT/US99/11913-   PCT Appln. PCT/US99/11913-   PCT Appln. PCT/US99/18441-   PCT Appln. WO 98/33791-   Peptide Synthesis, 1985-   Physicians Desk Reference.-   Pietrosemoli et al., J. Prot. Res., 6:3519-3526, 2007.-   Protective Groups in Organic Chemistry, 1973-   Protein NMR Spectroscopy, Principles and Practice, 1996-   Remington's Pharmaceutical Sciences, 15^(th) ed., 1035-1038 and    1570-1580, Mack Publishing Company, PA, 1980.-   Schafineister et al., J. Amer. Chem. Soc., 122(24):5891-5892, 2000.-   Solid Phase Peptide Synthelia, 1984-   Stelzer et al., J. Gen. Physiol., 128:261-272, 2006.-   The Merck Index, 11th Edition.-   Weber, Advances Protein Chem., 41:1-36, 1991.-   Wider, Bio Techniques, 29:1278-1294, 2000.-   Young et al., In: Handbook of Applied Therapeutics, 7.1-7.12 and    9.1-9.10, 1989.

1. A method of treating heart failure comprising: (a) identifying apatient exhibiting one or more symptons of heart failure; and (b)administering to said patient an inhibitor of the interaction of MyosinBinding Protein C (MyBP-C) and myosin.
 2. The method of claim 1, whereinsaid inhibitor is a peptide derived from the MyBP-C binding site formyosin. 3-12. (canceled)
 13. The method of claim 1, whereinadministering the inhibitor is performed intramuscularly, intravenouslyor by direct injection into cardiac tissue.
 14. The method of claim 1,wherein administering the inhibitor comprises oral, transdermal,sustained release, controlled release, delayed release, suppository, orsublingual administration.
 15. The method of claim 1, further comprisingadministering to said patient a second heart failure therapy.
 16. Themethod of claim 15, wherein said second therapy is selected from thegroup consisting of a beta blocker, an ionotrope, a diuretic, ACE-I, AIIantagonist, BNP, or a Ca⁺⁺ channel blocker.
 17. The method of claim 15,wherein said second therapy is administered at the same time as saidinhibitor of the interaction of MyBP-C and myosin.
 18. The method ofclaim 15, wherein said second therapy is administered either before orafter said inhibitor of the interaction of MyBP-C and myosin.
 19. Themethod of claim 1, wherein treating comprises improving one or moresymptoms of heart failure.
 20. The method of claim 19, wherein said oneor more improved symptoms comprises increased exercise capacity,increased cardiac ejection volume, increased cardiac ejection fraction,decreased left ventricular end diastolic pressure, decreased pulmonarycapillary wedge pressure, increased cardiac output, or cardiac index,lowered pulmonary artery pressures, decreased left ventricular endsystolic and diastolic dimensions, decreased left and right ventricularwall stress, decreased wall tension, increased quality of life, anddecreased disease-related morbidity or mortality.
 21. A method ofslowing the progression of heart failure comprising: (a) identifying apatient at risk of developing severe heart failure; and (b)administering to said patient an inhibitor of the interaction of MyosinBinding Protein C (MyBP-C) and myosin. 22-34. (canceled)
 35. The methodof claim 21, wherein the patient at risk may exhibit one or more of alist of risk factors comprising long-standing uncontrolled hypertension,uncorrected valvular disease, chronic angina, recent myocardialinfarction, congenital predisposition to heart disease or pathologicalhypertrophy.
 36. The method of claim 21, wherein the patient at risk maybe diagnosed as having a genetic predisposition to heart failure. 37.The method of claim 21, wherein the patient at risk may have a familialhistory of heart failure.
 38. The method of claim 21, further comprisingadministering to said patient a second heart failure therapy.
 39. Themethod of claim 38, wherein said second therapy is selected from thegroup consisting of a beta blocker, an ionotrope, a diuretic, ACE-I, AIIantagonist, BNP, or a Ca⁺⁺ channel blocker.
 40. The method of claim 21,wherein said subject is a heart transplant recipient. 41-45. (canceled)46. An isolated peptide comprising a MyBP-C binding site for myosin. 47.The peptide of claim 46, wherein said peptide is of no more than 50residues and comprise the sequence LKKRDXFRRD (SEQ ID NO: 1), where X isA, V or D.
 48. The peptide of claim 47, wherein the peptide comprisesthe sequence FSSLLKKRDXFRRD (SEQ ID NO: 48), FSSLLKKRDXFRRDXK (SEQ IDNO: 49), LKKRDXFRRDXKLE (SEQ ID NO: 50), SLLKKRDXFRRDXKLE (SEQ ID NO:51), or FSSLLKKRXAFRRDXKLE (SEQ ID NO: 54).
 49. The peptide of claim 47,wherein said peptide is no more than 25, 30, 35, 40 or 45 residues. 50.The peptide of claim 47, wherein said peptide comprises a cellpenetrating domain.
 51. The peptide of claim 47, wherein said peptidecomprises some or all D-amino acids.
 52. The peptide of claim 51,wherein said peptide comprises all D-amino acids and is in aretro-inverso configuration.
 53. A pharmaceutical composition comprisingan isolated peptide comprising a MyBP-C binding site for myosin.